P391: FLT3-ITD MEASUREMENT AT DIAGNOSIS AND FOR THE ASSESSMENT OF MINIMAL RESIDUAL DISEASE AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION IN PATIENTS WITH ACUTE MYELOID LEUKEMIA: CDNA VS. DNA
Authors: D. Carbonell, M. Chicano, A. Cardero-Illán, G. Rodriguez-Macías, P. Muñiz, R. Bailén, G. Oarbeascoa, I. Gómez-Centurión, J. Anguita, M. Kwon, J. L. Díez-Martín, I. Buño, C. Martínez-Laperche
Journal: HemaSphere (2022)

Background: DNA is standardly preferred to cDNA for the analysis of FLT3- internal tandem duplication (ITD). Although its screening is mandatory at diagnosis in clinical routine, its use as minimal residual disease (MRD) marker after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains controversial due to the poor sensitivity of its detection method and its apparent instability during the disease. Aims: To compare FLT3-ITD mutation analysis in DNA and cDNA samples at diagnosis and to prove the usefulness of its expression measurement as MRD marker after allo-HSCT. Methods: Forty-six diagnosis samples from 46 patients and 80 samples from 34 patients who underwent an allo-HSCT (34 pre-HSCT samples, 34 at day 30 after infusion and 12 before relapse) were analyzed. DNA was purified using a Maxwell RSC Blood DNA Kit (Promega), RNA was isolated using TRIzol reagent (Invitrogen) and cDNA synthesis was performed using the First Strand cDNA Synthesis kit (Roche). FLT3-ITD was analyzed in both DNA and cDNA samples by fragment analysis using an ABI3130xl DNA sequencer (Applied Biosystems). Fragment analysis was analyzed through Peak Scanner Software (Thermo Fisher). Allelic ratio (AR) quantification was performed by dividing the area under the curve of the mutant allele by the area under the curve of the wild type allele. Every clone was analyzed as an event, comparing its DNA ratio with its cDNA ratio. Results: Median AR of the 58 FLT3-ITD clones at diagnosis were 0.54 [0-9.47] in DNA samples and 0.63 [0.012-13] in cDNA samples, (Wilcoxon test, p<0.001, Figure A). In six patients, FLT3-ITD AR was <0.5 in DNA and ≥0.5 in cDNA, changing their prognosis based on ELN algorithm. One of them was not candidate for chemotherapy and of the five remaining, four were refractory or relapsed after intensive treatment. In terms of sensitivity, cDNA was better than DNA, quantifying higher AR values in most cases, optimizing the detection of minor clones and the prognostic classification. In respect of HSCT samples, seven patients were positive for FLT3-ITD mutation pre-HSCT in cDNA and two in DNA. Of the seven patients which were positive by cDNA, four relapsed after HSCT. The three patients who did not relapse had FLT3-ITD AR lower than 0.05. As for the two patients who tested positive in the DNA samples, only the one who also tested positive for cDNA relapsed. Despite the small sample size, these results reveal that the analysis of pre-HSCT cDNA samples could have a predictive value for relapse. No patient was positive on day 30 after HSCT, suggesting that it may not be useful, whether the DNA or cDNA sample is studied. However, the analysis at this time point could be crucial in those patients who may relapse early. On respect of the last sample before relapse (a median of 22 days before relapse, range 7-85), of the 12 cases, three were positive for FLT3-ITD mutation in DNA samples and nine were positive in cDNA samples. Great differences were observed between AR of both type of samples (Wilcoxon test, p<0.001, median 0 [0-0.04] vs. 0.15 [0-0.57], Figure B). In three patients FLT3-ITD cDNA measurement did not anticipate the relapse, due to the loss of the mutation or because the day on which the sample was analyzed was too early to detect the mutation, issues that can be solved by complementing with other MRD markers. Image:Summary/Conclusion: In conclusion, cDNA fragment analysis of FLT3-ITD by capillary electrophoresis is an easy-to-implement technique that could be a great alternative approach in AML patients at diagnosis and in allo-HSCT monitoring.